The effect of exercise and oxidant antioxidant intervention on the level of neurotrophins and free

73 Pages·2009·1.07 MB·English

The effects of exercise, oxidants and antioxidants
on neurotrophins and oxidative damage on spinal
cord of rats

Thesis

Savvas Siamilis

Semmelweis University
Doctor School for Sports Science
Sport and Educational Sciences

Supervisor: Dr. Zsolt Radak, D.Sc.

Opponents: Dr. Janos Feher, D.Sc.
Dr. Peter Ferdinandy, D.Sc.

Chairman of the Committee: Dr. Gabor Pavlik, D.Sc.
Members of the Committee: Dr. Robert Frenkl, D.Sc.
Dr. Janos Meszaros, C.Sc.
Dr. Jozsef Pucsok, D.Sc.

Budapest
2009
TABLE OF CONTENTS

List of Abbreviations........................................................................................................3
LIST OF FIGURES..........................................................................................................4
1 THE PROBLEM......................................................................................................6
1.1 Introduction......................................................................................................6
1.2 Hypotheses.....................................................................................................20
1.3 Objectives.......................................................................................................22
1.4 Delimitations of the study..............................................................................23
1.5 Limitations of the study..................................................................................23
1.6 Definition of terms.........................................................................................23
2 METHODS.............................................................................................................26
2.1 Animals...........................................................................................................26
2.2 Pilot study.......................................................................................................28
2.3 Instrumentation and collection of data (Biochemical assays and equipments)
29
2.3.1 Treadmill apparatus:..............................................................................29
2.3.2 Treadmill apparatus...............................................................................29
2.4 Independent variables.....................................................................................34
2.5 Dependent variables.......................................................................................35
2.6 Treatment of Data...........................................................................................35
2.7 Statistical analysis..........................................................................................35
3 ANALYSIS OF RESULTS....................................................................................37
3.1 Free Radical Accumulation (EPR Results):...................................................37
3.2 BDNF Protein Content:..................................................................................37
3.3 Free Radical accumulation and BDNF protein concentration:.......................38
3.4 Protein oxidative damage (Western blot):......................................................38
3.5 TrkB mRNA expression (RT-PCR data):.......................................................38
3.6 GDNF protein content:...................................................................................38
3.7 ERK1/2 Activation:........................................................................................39
4 DISCUSSION........................................................................................................48
4.1 Interpretation of Results.................................................................................48
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5 CONCLUSIONS....................................................................................................55
5.1 Conclusions....................................................................................................55
5.2 Recommendations for Further Research........................................................56
6 SUMMARY..........................................................................................................57
6.1 Summary.........................................................................................................57
6.2 Összefoglalás..................................................................................................59
Bibliographical data of most important publications.....................................................61
REFERENCES...............................................................................................................62
Acknowledgements........................................................................................................72

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List of Abbreviations

BDNF: brain derived neurotrophic factor
CNS: Central nervous system
CREB: Cyclic AMP response element-binding protein
EPR: Electron paramagnetic resonance
ERK1/1: extracellular signal kinase
GDNF: glial cell line derived neurotrophic factor
MAPK: mitogen activated protein kinase
mRNA: messenger ribonucleic acid
PBN: N-tert-butyl-a-phenyl nitrone
RCD: Reactive carbonyl derivatives
ROS: reactive oxygen species
RT-PCR: Real time polymerase chain reaction
TBARS: thiobarbituric reactive substances
TrkB: Tyrosine kinase receptor B

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LIST OF FIGURES AND TABLES

Figures (Introduction’s Section)
1. Oxygen flux through mitochondria at rest and ROS production 7
2. Mitochondrial complexes and superoxide radical production 7
3. Balance between production and removal of ROS 11
4. Marked increase in ROS production consumes endogenous antioxidants 12
5. Oxidative stress occurrence without increase in normal production of ROS 13
6. Worst case scenario for oxidative stress occurrence 13
7. Uncontrolled generation of ROS and cell’s macromolecules 14
8. Regular exercise drives biological systems to adaptive responses 16
9. Neurotrophins, their specific receptors, signal transduction and neuronal effects 17
10. Factors influencing Neurotrophin release from neurocells 19

Tables (Introduction’s Section)
1. Reactive Oxygen Species Relevant to Biological Systems 9
2. Biological Defense Against Oxidative Stress 10

Tables (Method’s Section)
1. Mean pre and post-treatment body-weight and standard deviation in grams 28
2. Difference in grams and in percentage of body-weight increment between pre and
post-treatment periods among non-exercised and exercised groups. 29

Figures containing photos (Method’s Section)
1. Treadmill Apparatus 17

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Figures (Results Section)
1. Free Radical Concentration 27
2. EPR Spectra 28
3. BDNF Protein Content 29
4. Correlation Matrix of Free Radicals and BDNF Content 30
5. Western Blot Assay of Protein Carbonyls 31
6. TrkB mRNA Expression 32
7. TrkB mRNA Expression in Percentage 33
8. GDNF Protein Content 34
9. Western Blot Assay of ERK1/2 and phosphor-ERK1/2 35

Figures (Discussion Section)
1. BDNF can function as both autocrine and paracrine agent 53
2. BDNF’s transportation bound to blood platelets 54

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1 THE PROBLEM

1.1 Introduction

Oxygen free radicals are products of normal cellular metabolism.
Mitochondrion, the organelle of the cell responsible for cellular respiration and energy
production is the major site of their production (figure 1.1.1 and figure 1.1.2). A free
radical is a molecule with one or more unpaired electrons in atomic or molecular
orbitals. The reactivity of free radicals results from their desire to attain an electron of
opposing spin direction. As a radical reacts with another molecule in order to pair with
another electron to be stabilized, a radical chain reaction begins. The radical chain
reaction continues until a termination reaction takes place. The termination reaction
occurs when radicals react with each other or with an antioxidant to produce non-radical
species (Radak, 2000, chap.1).
Radicals derived from oxygen represent the most important class of radical
species generated in living systems. Among free radicals, the typical ones are: super
oxide radical (O -), hydroxyl radical (HO.), nitric oxide radical (NO.) etc, (see table
2
1.1.3). Non radical species include among others: singlet oxygen (1O ), hydrogen
2
peroxide (H O ), ozone (O ) etc, (see table 1.1.3). Free radicals and non radical
2 2 3
derivatives of oxygen are collectively termed reactive oxygen species (ROS).

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Fig. 1.1.1 Oxygen flux through mitochondria at rest and ROS production

1.1.1 Under resting conditions oxygen flux through mitochondria produces
approximately 1-2% ROS.

Fig. 1.1.2 Mitochondrial complexes and superoxide radical production

1.1.2 The figure shows the major production side of superoxide radical (O .-) in
2
mitochondrial complexes 1 and 3. Immediately after production superoxide radical is
buffered by the antioxidant enzyme superoxide dismutase (SOD) and produces the less
reactive hydrogen peroxide (H O ).
2 2

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Table 1.1.3 Reactive Oxygen Species Relevant to Biological Systems

RReeaaccttiivvee OOxxyyggeenn MMoolleeccuullaarr
SSppeecciieess FFoorrmmuullaa
Free radical species
SSuuppeerrooxxiiddee rraaddiiccaall O -
2
HHyyddrrooppeerrooxxyyllrraaddiiccaall HOO.
HHyyddrrooxxyyll rraaddiiccaall HO.
NNiittrrooggeenn mmoonnooxxiiddee NO
AAllkkooxxyyllrraaddiiccaall RO.
PPeerrooxxyyllrraaddiiccaall ROO.
Nonradicalspecies
Singlet oxygen 1Δ O (1O )
g 2 2
Hydrogen peroxide H O
2 2
Peroxynitrite ONOO-
Hydroperoxide ROOH
Ozone O
3
Hypochlorousacid HC1O
Excited carbonyl RR’CO*

1.1.3 The table shows the free radical and non-radical species which are
collectively called reactive oxygen species (ROS). The table was adopted by (Radak,
2000, chap 1, p. 6).

ROS are well recognized for playing a dual role as both deleterious and
beneficial species, since they can be either harmful or advantageous to living systems
(Valko et al. 2006; Radak et al. 2004). The delicate balance between beneficial and
harmful effects of ROS is very important aspect of living organisms and is achieved by
mechanisms called ‘’redox regulation’’. The redox regulation machinery incorporates
enzymatic and non-enzymatic antioxidants as well as repair enzymes (table 1.1.4).

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Table 1.1.4 Biological Defense Against Oxidative Stress

Antioxidant Defence Occurrence
Preventive antioxidant enzymes
Superoxide dismutase Cu,Zn-SOD Cytosol
Ec-SOD Extracellular fluid
Mn-SOD Mitochondria
Fe-SOD Cytosol, chloroplsts
Catalase Peroxisomes
Glutathione peroxidase Cytosol, extracellular fluid, bio membranes
Glutathione S -transferase Cytosol
Antioxidants
α–Tocopherol(vitamin E) Biomembranes, lipoprotein
Ascorbic acid (vitamin C) Cytosol, extracellular fluid
Ubiquinol Biomembranes, lipoprotein
Carotenoids Biomembranes, lipoprotein
Reduced glutathione (GSH) Cytosol, extracellular fluid
Uric acid Cytosol, extracellular fluid
Oxidation damage repair Occurrence
Repair enzymes
Nuclei, cytosol
Nuclease
Nuclei, cytosol
Glycosylase
Cytosol
DNA polymerase
Cytosol
Protease
Biomembranes, lipoprotein
Phospholipase

1.1.4 The table shows enzymatic and non-enzymatic antioxidants as well as
oxidation damage repair enzymes along with their place of occurrence. Adopted by
(Radak, 2000, chap 1, p. 23).

Under normal conditions at rest and in healthy individuals the production of
ROS is compensated by the enzymatic and non enzymatic antioxidants (figure 1.1.5).

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